Water-soluble zinc pyruvates or their hydrates, method for the product ion thereof and their use

ABSTRACT

Water-soluble zinc pyruvates and hydrates thereof of the formula I                    
     where x=1.8 to 2.2 and n=0 to 5 are described. The inventive zinc pyruvates are distinguished by a high purity and excellent storage stability and thermal stability and can be employed especially for use as therapeutic agent for treating diabetes, cold prophylaxis, virus inhibition, cytoprotection, as microbicide and as radical absorber and as food supplement or supplement for the prophylaxis and prevention of zinc deficiency symptoms.

The present invention relates to water-soluble zinc pyruvates andhydrates thereof having a high purity and good storage stability andthermal stability, a process for their preparation and use thereof.

It is known that salts of pyruvic acid, called pyruvates, have valuablephysiological, therapeutic and dietary properties. Pyruvates are used toincrease stamina and strength in the sports sector, for weight reductionand body fat reduction and as a protective substance for body cells andbody tissue, particularly cardiovascular, hepatic, nephrotic, peritonealand neuronal tissue, and as antioxidant, both as a substance forinhibiting radical formation and also as a radical absorber substance inbody cells, body tissues and cells of the synovial tissue, in the healthsector and as food supplement agent. In addition, pyruvates are used aswound healing preparations, for treating diabetes because of theiraction in reducing blood sugar and for treating kidney diseases(acutekidney failure, kidney stones suffering).

Furthermore, it has been known for over 100 years that zinc is anessential trace element for plants, animals and humans. The occurrenceof a zinc deficiency in humans has been thought to be improbable for along time. Therefore, the interest of dietetics in this trace elementwas initially low. In the 1950s, it was then demonstrated that zincdeficiency can occur in humans with typical symptoms. The research wasintensified worldwide as in 1961 the endemic occurrence of hypogonadismand dwarfism in Iran and the Nile delta of Egypt could be refered to azinc deficiency. Routine analytical measurements in foods and tissuesamples contributed to a better understanding of the role of the traceelement zinc in biochemical processes. Currently, the clinical symptomsof a severe zinc deficiency are known, but the pathological changesoccurring in the zinc deficiency cannot be explained till now.

Zinc is a constituent of metalloenzymes and stabilizes organicstructures and membranes. Over 200 different zinc-dependent enzymes havenow been identified. Superoxide dismutase, alkaline phosphatase, DNApolymerase and RNA polymerase and carboxypeptidase are examples ofthese. Zinc participates in nucleic acid synthesis, protein metabolism,lipid metabolism, carbohydrate metabolism, bone metabolism, oxygentransport, dark adaptation and the antioxidative protective system ofthe human body. Zinc is essential for all forms of life because it playsan important role in transcription and translation and expression ofgenes. Some enzymes contain zinc in the active center, where it acts aselectron acceptor. In other enzymes and non-enzyme proteins and innucleic acids zinc has structural and stabilizing functions (zincfinger). In the pancreas zinc participates in insulin synthesis andinsulin storage. In addition, it fulfills important tasks in the immunesystem.

In the case of zinc, in addition to the absolute content, thebioavailability is also critical for the importance of a food as a zincsource. Absorption of zinc from foods of animal origin is generallyhigher than from foods of plant origin. Zinc, which forms poorlyabsorbable complexes with phytic acid, has low availability fromphytate-rich foods (for example whole kernel corn). Simultaneouslyincreased consumption of the metals iron, copper, tin and cadmium alsoinhibit absorption. The presence of amino acids, peptides and organicacids increases bioavailability. Muscle meat, milk products, fish andparticularly shellfish (for example oysters up to 160 mg of Zn/100 g)are good zinc sources. Dark meat (beef: 4.3 mg of Zn/100 g) has a higherzinc content than light meat (chicken: 1.0 mg of Zn/100 g). Most fruitand vegetables make only a small contribution to the zinc supply. Thezinc content of cereal products is greatly dependent on the degree ofprocessing, since zinc is predominantly localized in the outer layers ofthe grain. The mean absorption rate of zinc from different types ofbread is around 10%, whereas this can be up to 40% from meat fromvarious types of poultry.

Zinc is partly released from the bound form in foods by digestiveenzymes and gastric acid, and binds loosely to low-molecular-weightligands such as amino acids, peptides, organic acids and phosphates,which is of importance for the extent of zinc absorption. Zinc isabsorbed by a saturable carrier process in the duodenum and jejunum.Absorbed zinc in blood plasma is bound to albumins, a₂-macroglobulin andtransferrin and transported. The proportion of zinc bound to plasmaproteins however makes up only approximately 20% of the zinc content inthe blood. The greatest proportion (75%) is present as constituent ofcarboanhydratase in the erythrocytes. A further 3% is present asconstituent of alkaline phosphatase in leukocytes.

Zinc is present in all body tissues and body liquids in differingconcentrations. Body levels in adults are in total 2-3 g of zinc.Approximately 60% is present in skeletal musculature and 30% in thebones. Zinc is primarily an intracellular ion which occurs at onlyrelatively low concentrations in extracellular fluids. Only 0.1% of thetotal body zinc circulates in the plasma. There zinc contents aresubject to strict homeostatic control. Humans have to have a regularzinc uptake from food. Zinc is predominantly excreted via the intestine.It is lost here together with digestive secretions and sloughedintestinal epithelial cells. Endogenous losses can be 2 to 4 mg/day. Afurther 0.5 mg each are excreted with the urine and via the skin (skinscales, hair and sweat). In inflammatory intestinal and kidney diseases,and also in the case of alcoholism, excretion is significantlyincreased.

Severe forms of zinc deficiency have been observed in humans havingcongenital disturbances of zinc metabolism (acrodermatitisenteropathica), in patients having inadequate parenteral nutrition andin patients with Morbus-Crohn's disease. The clinical pattern ofsymptoms includes dermatitis at the ends of limbs and around the mouth,diarrhea, loss of appetite, hair loss and neuropsychiatric disturbances,thrieving depression and growth depression, increased susceptibility toinfections and delayed wound healing and disturbances of sexualdevelopment.

Furthermore, zinc can be used to prevent the progressive impairment ofglucose tolerance with age. With increasing age, independently of bodyweight, there is a highly significant impairment in glucose toleranceand a significant decrease in insulin secretion after administration ofglucose. The basal serum insulin values decrease with increasing age. Areduction in glucose conversion rates with age has been found forglucose metabolism of the brain. Animal experiments and clinical studiesindicate that zinc deficiency plays an integrating role in theprogressive impairment of age-related glucose tolerance.

Zinc is particularly suitable for treating diabetes mellitus. Zincimproves glucose tolerance and can markedly increase insulin action.Zinc is an important therapeutic for diabetics. According to the GermanDiabetes Society (Deutsche Diabetes-Gesellschaft, DDG), there currentlylive in Germany approximately 4 million diabetics (5% type I diabetics,95% type II diabetics).

According to estimates of leading diabetologists, this figure willdouble by the year 2000.

Hyperglycemia characterizes diabetes mellitus as a cardinal symptom. Indiabetics there is either an insulin deficiency and/or an insulinresistance. Insulin regulates the blood glucose level. Trace elementscannot cure diabetics or act as therapies alone. Despite this, theessential trace element zinc plays a critical role in regulation ofblood sugar. With zinc deficiency pathological glucose tolerancefrequently occurs, which can be normalized alone by regular zincadministration.

The essential trace element zinc improves the insulin storage in type IIdiabetics. Zinc deficiency inhibits insulin activity and insulinreceptor formation. The daily zinc requirement, according to the GermanNutrition Society (Deutsche Gesellschaft für Ernährung, DGE), in adultsis from 12 to 15 milligrams. The average zinc intake via foods(particularly in oysters, innards, meat) is somewhat below thisrecommendation. Diabetics belong to zinc supply risk groups, since theirzinc losses via urine are markedly increased. The level of zincexcretion increases with the extent of glucosuria (sugar excretion viaurine). This relates especially to diabetics suffering from diabeticnephropathy. The McNair study shows urine zinc excretions higher by 50to 150% in diabetics compared with healthy persons. If urine sugarvalues are positive, the zinc loss is much higher and can exceed 2 to 3times the norm. In type II diabetics, Perger and coworkers, afteradministration of zinc for 6 weeks, found a decrease in the averagefasting blood sugar values from 250 to 142 mg %. In other studies, boththe fasting and also postprandial blood sugar level decreases markedlywith zinc administration. Therefore the daily intake of from 15 to 30milligrams of zinc via tablets appears useful.

With zinc administration, wounds heal better, for example in the case ofwhat is termed diabetic foot on the base of poor blood sugar values,peripheral occlusive disease and diabetic polyneuropathy. This is due,inter alia, to the antiinflammatory effect of zinc.

The exact human zinc requirement is unknown. According to balancestudies, the daily obligatory zinc losses which must be replaced are 2.5mg. At an average bioavailability of 20%, this gives for Germany arecommended daily intake of 15 mg for men. Because of the lower averagebodyweight, a zinc intake for women of 12 mg/day is recommended. In theUSA the recommended daily intake (RDI) for adults is 15 mg of zinc.

In the literature, zinc pyruvate has only been described to date once byJ. J. Berzelius, Annalen der Physik und Chemie (1835) 36, 20, as a zincoxide salt of pyruvic acid. Beilstein, Volume 3 of the main series, page612, cites this reference, specifying zinc pyruvate as trihydrate.

Berzelius produced the salt either by transforming basic zinc carbonateor metallic zinc with dilute pyruvic acid. The resultant salt, accordingto Berzelius, is a sparingly soluble powder. However, followingBerzelius's instructions for synthesizing zinc pyruvate gives asparingly soluble powder which, by modern analytical testing methods(NMR, IR, HPLC), has been unambiguously identified as zinc parapyruvate.

It has also been found that the previously known process for producingso-called “zinc pyruvates” by neutralizing pyruvic acid with zinccarbonate, zinc hydroxide or zinc oxide and the reaction of zinc metalwith pyruvic acid leads to a sparingly soluble zinc parapyruvate withlarge amounts of byproducts, since pyruvic acid and the pyruvate ionsreact via aldol addition reactions or aldol condensation reactions toacyclic or cyclic dimers and polymer s of pyruvic acid, respectively.

Zinc parapyruvates contaminated in this way are unsuitable for use astherapeutics and furthermore are not sufficiently storage-stable,because during storage, inter alia, dimeric, polymeric and cycliccompounds a re formed, which can be toxic.

Acyclic compounds which may be mentioned are parapyruvic acid(4-hydroxy-4-methyl-2-oxoglutaric acid) and its salts, and the higheraldol addition products. In addition, oxalic acid and methylsuccinicacid can be formed as byproducts. The acyclic pyruvic acid polymers canform via lactonization reactions, ketalization reactions and otherreactions cyclic compounds, for example 2-hydroxy-2-methyl-4-oxoglutaricacid -5-lactone, trimesic acid derivatives, isophthalic acid derivativesand pyrantricarboxylic acid derivatives (reference: Beilstein, BasicSeries Volume 3, pp. 608-613; 1st Supplementary Series, pp. 217-219; 2ndSupplementary Series, pp. 393-401; 3rd Supplementary Series, pp.1146-1156; 4th Supplementary Series, pp. 1505-1510). These byproductsoccur in the same manner in the storage of zinc pyruvates which areprepared by the previously known process. These abovementioned acyclicand cyclic byproducts and decomposition products of pyruvic acid and itssalts can be physiologically incompatible or toxic.

The object therefore underlying the present invention was to developwater-soluble and storage-stable zinc pyruvates which do not have thementioned disadvantages corresponding to the prior art, but have a highpurity and are therefore physiologically safe.

This object was achieved according to the invention by preparing thezinc pyruvates and hydrates thereof of the formula I

where x=1.8 to 2.2 and n=0 to 5.

This is because it has surprisingly been found that the inventive zincpyruvates have a high purity and are virtually free of any toxicbyproducts and have an excellent storage stability and thermalstability.

This was surprising, because in the inventive preparation, pyruvic aciddoes not undergo, or undergoes to a very limited extent, condensationreactions and decomposition reactions with formation of aldol adducts,although transition metals such as zinc should catalyze thepolymerization of pyruvic acid.

The inventive zinc pyruvates which have high water solubility and can beunambiguously characterized by elemental analysis, IR spectroscopy, NMRspectroscopy and by HPLC content determination, contain the zinc cationand the pyruvate anion, which can be present as oxopropionate and/or2,2-dihydroxypropionate anion, in a molar ratio of from 1.8 to 2.2:1 andpreferably in the stoichiometric ratio of 2:1.

In addition, the inventive zinc pyruvates are present either completelyanhydrous or in the form of hydrates, where n can be from 0 to 5 andpreferably from 0 to 3. According to a preferred embodiment, the zincpyruvates are present in a solid microcrystalline form which isdistinguished by a particularly high storage stability.

The zinc pyruvate content can thus be from 72.7 to 100% and the crystalwater content from 0 to 27.3% (equivalent to from 0 to 5 mol of crystalwater). Based on the anhydrous substance, the zinc pyruvate content isfrom 99 to 100%.

The inventive zinc pyruvates are prepared by reacting zinc salts oforganic acids or acidic organic keto compounds or hydroxyl compoundswith pyruvic acid in the temperature range from −20 to +90° C., in thepresence or absence of a solvent or diluent. Preferably, the reaction iscarried out at from 10 to 50° C.

The organic acid used can in principle be any physiologically safecarboxylic acid which may optionally be substituted with amino, keto orhydroxyl groups. Preferably, the organic acid or acidic organic ketocompound or hydroxyl compound used is an acid selected from the groupconsisting of formic acid, acetic acid, propionic acid, butyric acid,lactic acid, ascorbic acid, citric acid, tartaric acid, succinic acid,maleic acid, fumaric acid, malic acid, aspartic acid, benzoic acid,gluconic acid, isovaleric acid, oleic acid, glycine or lysine.

These zinc salts of the organic acids or acidic organic keto compoundsor hydroxyl compounds can be used in anhydrous form, as hydrates or asmoist products.

The pyruvic acid can also be used in the inventively proposed processoptionally as free acid, as aqueous solution or dissolved in a solventor diluent. Suitable solvents or diluents in this case are preferablywater and/or organic solvents, for example alcohols (methanol, ethanol,isopropanol, cyclohexanol), ethers (diethyl ether, tetrahydrofuran,1,4-dioxane), ketones (acetone, methyl ethyl ketone, cyclohexanone),esters (methyl acetate, ethyl acetate, ethyl formate), organic acids(formic, acetic, propionic, lactic, pyruvic acid) and aliphatichydrocarbons (pentane, hexane, cyclohexane) and aromatic hydrocarbons(toluene).

However, the organic zinc salts can also be reacted with pyruvic acid assuch in the absence of solvent or diluent.

According to a preferred embodiment, it is also possible in the contextof the present invention to generate pyruvic acid as an intermediate,that is to say, for example, by reacting alkali metal pyruvate, forexample sodium pyruvate or potassium pyruvate, with an inorganic acid,for example sulfuric acid or hydrochloric acid, in the temperature rangefrom −20 to +90° C., preferably from −10 to +60° C.

The ratio of organic zinc salt to pyruvic acid can be varied withinbroad limits, but it has proved to be particularly advantageous to reactthe organic zinc salts and pyruvic acid in stoichiometric (2:1) orapproximately stoichiometric ratios (1.8 to 2.2:1).

The procedure of the inventive process is substantially unproblematic,and it can be performed by customary methods and in known processingapparatuses such as in kneaders, mixers, blade dryers and stirredvessels. In this manner zinc pyruvates are obtained without furtherworkup steps in high yield and purity.

The inventive zinc pyruvates have valuable biological and medicalproperties and are used as therapeutics for treating diabetes, coldprophylaxis, virus inhibition, cytoprotection, as microbicide and asfree-radical scavenger or as food supplement or as supplement for theprophylaxis and prevention of zinc deficiency syndrome, such as growthdisorders in children and adolescents, weight loss, increasedsusceptibility to infections, poor wound healing, taste and odordisorders, delayed puberty, decrease of appetite, vision disorders,diarrhea, skin diseases, hair loss and emotional disorders.

The inventive zinc pyruvates surprisingly show antiviral properties, inparticular against influenza A and influenza B viruses, herpes I, II andIII viruses and the rhinoviruses, antimicrobiological activities againstbacterial infections, for example Pseudomonas immunofluorescens,Pseudomonas aerogenes strains, staphylococci and streptococci andcausative agents of sinusitis, tonsilitis and catarrhal inflammationsboth of virucidal or bacterial genesis, pyoderma and boils andrefractory antiseptic wound treatments. The antimycotic activitiesrelate in particular to dermatomycoses, mycoses of general types causedby yeasts, molds, dermatophytes, candidoses and yeasts and alsopityriasis versiculor. This also relates to prophylaxis of the skin andmucosa, in particular in the case of postoperational surgical woundtreatment in the case of (possibly occurring) bacterial wound infectionsand secondary infections in the dermatological area, where they arecaused by Gram-positive and/or Gram-negative meclocyclin-sensitivemicroorganisms.

The inhibition of influenza A and B viruses by zinc pyruvates wasdemonstrated experimentally by inhibition of influenza A- and B-inducedviral hemagglutination (vHA) of erythrocytes (RBC) and in vitro in cellcultures by the inhibition of viral infectivity. By comparing thedetermined inhibition constants of the virus-induced infectivity (plaquereduction, PFU=plaque forming units) in Madin-Daby-Canine-Kidney (MDCK)cells, the cytotoxicity of zinc pyruvate, and the vHA at the level ofIC₅₀ concentrations, it was possible to demonstrate a) the potentiationof the antiviral action as exemplified by IC₅₀ or vHA and reduction ofinfectivity by zinc pyruvate; b) the cytoprotective action of zincpyruvate at concentrations at which not only pyruvate but also zincsalts (inorganic and organic) demonstrate no biological activity,especially inhibitory activities, and c) the therapeutic index of zincpyruvate (2000) which is high owing to its very low cytotoxicity, and avery high in vitro selectivity ratio for zinc pyruvate which existsneither for pyruvate nor for zinc salts (zinc ions). The sametherapeutic index could not be achieved by administering the twocomponents, such as Zn²⁺ions and pyruvate, alone or as a loosecombination, or in a sequential manner. In this case the therapeuticindex even falls to 340, if, for example, first pyruvate and then Zn²⁺isadded to the cell cultures, in the reverse case (first Zn²⁺, thenpyruvate) even to 250. Therefore, administering the inventive zincpyruvate has a synergistic action which leads to significant advantagescompared with administration in the form of the individual componentszinc and pyruvate.

A further synergistic effect of zinc pyruvate is the reduction of activeoxygen radicals and/or hydroxyl radicals, in particular in inflammatoryprocesses. Thus, faulty lytic reactions in which both aggressive oxygenradicals and nitrogen oxide radicals participate, originating aspathogenic mechanisms in inflammatory diseases, are inhibited by zincpyruvate. For example, inter alia, the cytotoxic action of the secondaryproducts of these aggressive radicals is inhibited by zinc pyruvate and,inter alia, thus depolymerization of hyaluronic acid, proteoglycans,collagen fibrils, cytoskeletons and adhesin proteins and mucous andmembranous tissue is also suppressed or a temporary cell protection iscaused, as a result of which the infiltration of aggressive freeradicals can be prevented.

Circulating monocytes which, in the absence of inflammatory stimulants,penetrate the tissue and thus become resident macrophages, afteractivation, for example by superoxides (hydroperoxides), NO and O₂radicals and by inflammatory events, play a great role in the activationof enzymes, growth factors, cytokines and lymphokines. Lymphokines arereduced at inflammatory points in order to induce defense mechanismsagainst the invasion of bacterial or viral influences (LPS,hemagglutinin, neuraminidase, laminins, s-ICAM). Zinc pyruvate supportsor triggers the stimulation of the host-defense function by secretinginterferon-γ (IFN-γ) and thus plays a regulatory part in the secretoryactivity and humoral defense via the biosynthesis of IL-4 and IL-10.Especially the regulatory and in part inhibitory effect of zinc pyruvateon the release of HO₂ ^(∪) was demonstrated by the experimentalmeasurement of respiratory burst activity quantitatively under theinfluence of zinc pyruvate.

Finally, the invention relates to a process for treating disordersselected from the group consisting of diabetes, colds, viral andmicrobial infections and zinc deficiency and to a process for scavengingradicals or for cytoprotection, in which an effective amount of aninventive zinc pyruvate is administered to the patient to be treated,which can be a human or an animal.

The zinc pyruvate is preferably administered orally. However, otherforms of administration are conceivable, for example parenteral ortopical adminstration. The amount of zinc pyruvate administered can,because of the low toxicity, be varied within broad ranges. Thetreatment duration depends on the type and severity of the disease to betreated. However, the inventive preparations can be administered withoutdamage to health over a period of several weeks to months.

Administration is preferably performed in the form of a pharmaceuticalcomposition which comprises as active compound a water-soluble zincpyruvate, in particular a zinc pyruvate as defined above as activecompound if appropriate together with pharmacologically safe excipients,diluents and aids. In addition, obviously, the composition can compriseother pharmaceutical active compounds which are used together with thezinc pyruvate to treat a disease. For example, daily doses of from 0.01to 25 mg per kg of bodyweight of the treated patient are possible, whichcorresponds to supplying a total daily amount of from 0.7 to 1750 mg fora person weighing 70 kg.

The examples below are intended to illustrate the invention in moredetail.

EXAMPLES A. Preparation Examples Example A.1

To a solution of 88 g (1 mol) of pure pyruvic acid (99% pure) in amixture of 500 ml of ethyl acetate and 1000 ml of glacial acetic acidare added at 20° C. in the course of 30 minutes 105 g (0.48 mol) of zincacetate dihydrate and the mixture is stirred for 3 hours. Finally, thezinc pyruvate is filtered off with suction and washed with 2×250 ml ofethyl acetate. The yield of zinc pyruvate monohydrate is 122 g (95% oftheory).

(C₃H₃O₃)₂Zn·1H₂O, calculated: C 27.99%, H 3.13%, Zn 25.39%; found: C28.09%, H 3.28%, Zn 25.44%; m.p.>300° C.; IR (KBr) [1/cm]: 644, 737,854, 1190, 1350, 1372, 1412, 1650, 1703, 3222; ¹H-NMR (D₂O, 300 MHz):δ=2.42 (s, 3H, CH₃—CO), 1.54 (s, 3H, CH₃—C(OH)₂); HPLC contents (zincpyruvate): 92.8% =99.8% zinc pyruvate monohydrate.

Example A.2

To 455 g (5 mol) of 98.7% pure pyruvic acid are added at 40° C. in thecourse of 1 hour 55 g (0.25 mol) of zinc acetate dihydrate. The mixtureis stirred at 40° C. for a further 3 hours. After cooling to 15° C., themixture is stirred for 1 hour. Finally, the zinc pyruvate is filteredoff with suction, washed with 2×100 ml of ethyl acetate and dried at 50°C. and 15 mbar. The yield of zinc pyruvate monohydrate is 61 g (95% oftheory).

Example A.3

At a temperature of from 15 to 20° C., to a suspension of 110 g (1 mol)of sodium pyruvate in 200 ml of ethyl acetate 64.3 g (0.49 mol) of 70%strength sulfuric acid are added dropwise over a period of 45 minutes.After 3 hours the precipitated sodium sulfate is filtered off withsuction and the residue washed with 2×40 ml of ethyl acetate. To thefiltrate are added 250 g of concentrated acetic acid. The mixture isheated to 35° C. In the course of 30 minutes, 96.7 g (0.48 mol) of zincacetate monohydrate are introduced. The suspension is stirred furtherfor 3 hours. Finally, the zinc pyruvate is filtered off with suction andwashed with 2×100 ml of ethyl acetate. The product is dried to constantweight at 50° C. in a vacuum drying cabinet. The yield of zinc pyruvatemonohydrate is 124 g (96% of theory).

Example A.4

To a solution of 45.5 g (0.5 mol) of 98.7% pure pyruvic acid in 200 mlof glacial acetic acid and 200 g of ethyl acetate are added 20 g ofwater and 38.9 g (0.25 mol) of zinc formate are introduced at 40° C. inthe course of 1 hour. The mixture is stirred for 3 hours at thistemperature. After cooling to 15° C. the mixture is stirred for afurther 1 hour. Finally, the zinc pyruvate is filtered off with suction,washed with 2×100 ml of ethyl acetate and dried at 50° C. and 15 mbar.The yield of zinc pyruvate trihydrate is 71.4 g (97% of theory)

(C₃H₃O₃)₂Zn·3H₂O, calculated: C 24.55%, H 4.12%, Zn 22.27%; found: C24.64%, H 4.18%, Zn 22.52%; m.p.>300° C.; IR (KBr) [1/cm]: 644, 737,854, 1190, 1350, 1372, 1412, 1650, 1703, 3222; ¹H—NMR (D₂O, 300 MHz):δ=2.42 (s, 3H, CH₃—CO), 1.54 (s, 3H, CH₃—C(OH)₂); HPLC contents (zincpyruvate): 81.5% =99.8% zinc pyruvate trihydrate.

Example A.5 (Comparison)

Repetition of the instructions for preparing the zinc oxide salt ofpyruvic acid of J. J. Berzelius, Annalen der Physik und Chemie (1835)36, 20.

In 105.6 g (0.6 mol) of 50% strength aqueous pyruvic acid are dissolved34.2 g (0.1 mol) of zinc hydroxide carbonate hydrate (Aldrich 36,720-6). After the end of carbon dioxide evolution, the mixture isstirred for a further 2 hours. Finally, the resultant white precipitateis filtered off with suction, washed with 50 ml of water and 50 ml ofmethanol and dried at 50° C. and 15 mbar. The analyses clearlycharacterized the precipitate as zinc parapyruvate. The yield of zincparapyruvate trihydrate is 79.1 g (80% of theory).

(C₆H₆O₆)Zn·3H₂O, calculated: C 24.55S, H 4.12%, Zn 22.27%; found: C24.64%, H 4.18%, Zn 22.52%; m.p.>300° C.; IR (KBr) [1/cm]: 642, 669,798, 1075, 1113, 1140, 1205, 1228, 1342, 1396, 1474, 1609, 1677, 1713,3408; ¹H-NMR (D₆—DMSO, 300 MHz): δ=1.27 (s, 3H, CH₃—CO), 2.99 (d, 1H,CH₂), 3.25 (d, 1H, CH₂); HPLC contents (zinc parapyruvate): 81.5% =99.8%zinc parapyruvate trihydrate.

Example A.6 (Comparison)

In 35.2 g (0.2 mol) of 50% strength aqueous pyruvic acid are dissolved6.5 g (0.1 mol) of metallic zinc powder. After the completion ofhydrogen evolution, the mixture is stirred for a further 2 hours.Finally, the resultant white precipitate is filtered off with suction,washed with 50 ml of water and 50 ml of methanol and dried at 50° C. and15 mbar. The analyses clearly characterized the precipitate as zincparapyruvate. The yield of zinc parapyruvate tetrahydrate is 24.0 g (77%of theory).

B. Use Examples Example B.1 Assays for Macrophage Activation andCytokines

A modified assay was carried out according to Barbior et al. (B. M.Barbior, R. S. Kipnes, J. T. Curnutte, J. Clin. Invest., 52, 741, 1973).In this assay 5×10⁵ macrophages per titer and well were used in thepresence of 10% fetal calf serum with increasing concentrations of zincpyruvate (0-0.1-1×10³ mM). The introduced number of macrophage cellscorresponds to the release of HO₂ ^(∪) being, or ensures that it is,exactly proportional to the concentration of the cell count. The blankvalue (blank, only buffer) and a negative control and a positive control(authentic substance, zinc pyruvate) were carried out such thatspecificity of the superoxide anions was ensured. Thus the wells wereinoculated with 15 μg of superoxide dismutase at a cell count of 3.0×10⁵in order to act as a negative control.

The macrophages were activated either with phorbol myristic ester (20μg/ml) or zymosan (100 μg/ml). The macrophages thus stimulated werepretreated as negative control, blank or with the appropriateconcentration of zinc pyruvate (authentic compound) (determination ofcytoprotection) or added directly to the assay (determination ofinhibition), then washed with 0.02 M NaH₂PO₄ buffer (pH 6.5, 20° C.) andthen incubated with 0.5 ml of reaction mixture/Hank's solution (phenolred-free), 80 μM of ferricytochrome C (Sigma type IV) and 5 mM NaN₃,which acts as a cytochrome oxidase inhibitor, and with stimulant (≈50-70μl) at 37° C. for 20 min. Cytochrome C reduction was measured by thechange in extinction at 550 nm. The superoxide anion concentration wasdetermined via the difference in absorption at 550 nm in the presence orabsence of superoxide dismutase using the experimentally determined(working) extinction coefficient of 18.95/mM/cm (reduced or oxidized).The biochemical activities of inhibition of oxygen free radicalformation were measured and evaluated in accordance withMichaelis-Menten kinetics.

Inhibition of the release of superoxide radicals (anions) in thepresence of zinc pyruvate gave an inhibition constant of K_(i)=550±25 mMat an association constant of zinc pyruvate to the macrophages underthese in-vitro conditions of 150 nM. When the macrophages werepretreated with 1.0 mM zinc pyruvate for a period of 10 min at 37° C.and subsequently stimulated with phorbol myristic ester under the sameassay conditions, inhibition constants of K_(i(1))=150±25 mM andK_(i(1))=670±55 mM were found, respectively. The two differentinhibition constants are explained via the allosteric inhibition of thepretreated macrophages with respect to the release of HO₂ ^(∪) byalready inhibited macrophages which are no longer available to the assayor have become refractory (allosteric inhibition). The binding constantadditionally determined by equilibrium dialysis of only 150 nM zincpyruvate to the macrophages at a cell count of 10⁵ also verifies theunusual allosteric course of inhibition in the presence of zincpyruvate. Despite the increase in the concentration of the stimulant,although under saturated conditions (zinc pyruvate/macrophage), neitherthe binding constants for zinc pyruvate and that of the macrophages, northe inhibition constants K_(i) changed, but more oxygen radicals wereinactivated per unit of time. This meant that in the presence of zincpyruvate the turnover number of the macrophages to destroy the activeand aggressive oxygen radicals increased from ≅350 to ≅7100.

Example B. 2 Assay of Inhibition of NO Secretion

The assay of the effect of nitrogen oxide secretion by zinc pyruvate wasperformed using the GRIESS reaction (A. H. Ding, C. F. Nathan & D. J.Stuehr, J. Immunol., 141, 2407, 1988). The modified GRIESS reagent wascomposed as follows: 1 ml of 0.5% (g/g)naphthaleneethylenediamine·H₃PO₄, 1 ml of 0.5% (g/g) sulfanilacidamidein 1% (g/g) H₃PO₄. In order to activate the NO burst for macrophages,the macrophages (cell count: 2.0×10⁵) were incubated for 2 hours at 37°C. in 500 μl of GRIESS reagent by adding 75 units/ml of IFN-γ. Afteraddition of 20 μg/ml of lipopolysaccharide (LPS, E. coli, MRE 600), theNO determination was initiated and started, respectively. The mixturewas incubated for 12 hours at 37° C. under a stream of N₂. As negativeor positive control in the NO biosynthesis, 200 μg/ml ofN⁶-monoethylarginine were added together with LPS. The supernatant wasseparated off from the cells by centrifugation, GRIESS reagent was addedto the appropriate volume and then the extinction was measured after 5min at 525 nm (glass cuvette, 1 cm path length, 25° C.).

The inhibition constants for zinc pyruvate which were determined wereK_(i)=600±50 mM, and with pretreatment of the macrophages with zincpyruvate K_(i)=230±60 mM, although with only one Michaelis-Menteninhibition constant. That is to say, via the concentrationdependentinhibition of the active, aggressive and cytotoxic oxygen radicals andhydroperoxides (according to pH) and the cytosolic activation of thecells by zinc pyruvate, both the humoral and the cellular activationcomponents including the MHC III complex on the macrophage mannosylreceptor are modulated. This modulation also includes IL-1, IL-6 and theTNF-α factors. These elements occupy especially the pro-inflammatorypositions with corresponding reduction of the pro-inflammatory cytokinesecretion by zinc pyruvate. In this case, although the macrophages, inthe presence of zinc pyruvate, and in contrast to IFN-γ, IL-10, IL-4 andIL-13, are in the activated state, this is in such a manner that theycan stimulate the necessary protective cytokines, such as IL-1, IL-6 andTNF-α and can thus ensure protection from LPS, bacterial products(fever, edema, release of prostaglandins and possibly leukotrienes, SLSproducts) and also from viral products (increase in autoimmuneactivities). Thus the zinc pyruvate, via cellular induction, makes acontribution toward reducing the inflammatory events, by stimulating theantagonists of the pro-inflammatory cytokines.

Example B.3 In-vitro Inhibition of Viral Hemagglutination (vHAI) andInfectivity by Reducing Plaques in the Presence of Zinc Pyruvate

As mentioned above, the antiviral properties of zinc pyruvates weretested on epithelial MDCK cells for the influenza strainsA/Chile/1/83/or influenza B/Singapore/27/79/. The assay conditionscorresponded to those of K. Tabita, A. Sugiura, C. Enomoto, M. Furuyama,Med. Microbiol. Immunol., 165, 9-14, 1975. The infected MDCK cells weretreated for 30 min with zinc pyruvate and compared with cell cultureswhich had not been infected with influenza virus. A qualitative andquantitative control of the advancing infection was additionallyperformed by determining the viral proteins and the count of infectiousparticles in the cell culture supernatant.

The cell cytotoxicity was determined correspondingly under identicalculture conditions with identical but uninfected MDCK cells. Thevalidity of the results obtained was determined on the basis of the 95%confidence limit using the t-test values for the correspondingconfidence ranges.

The inhibition of influenza-induced hemagglutination (vHAI) oferythrocytes (RBC) was carried out in accordance with the protocol of G.N. Rogers, T. Pritchett, J. L. Lane, J. C. Paulsen, Virology, 131,394-408, 1983. The results are summarized in Table 1.

The low cytotoxicity of zinc pyruvate is notable and surprising. Thelysis of RBC cells begins at 1000 μg/ml, that is considerably higherthan for the inhibitory constants determined in vHAI, which are of theorder of magnitude of 50 μg/ml. The LD₅₀ concentration for the MDCKcells is on average from 800 to 1000 μg/ml for zinc pyruvate. That is tosay all of the values found are far above those of the biochemicallyactive inhibitor concentrations, so that the therapeutic concentrationsof zinc pyruvate in no manner exert a cytotoxic effect on the cells invitro, and for which reason the measured inhibition of the individualparameters is due to a specific interaction with the cellular apparatusor the virus and not to the lysis of the MDCK cells.

TABLE 1 Hemagglutination inhibition (HAI), plaque reduction assay of Znpyruvate HAI, μm Potency Plaque Reduction^(a) No. Inhibitor IC₅₀ HAI μM% 1 Zn(Ac)₂ 150.0 6.0 1700 15 2 Zn(Py)₂ 25.3 19.9 90.5 67 3 ZnCl₂ 100.05.0 1200 20 4 ZnO.H₂O ≈200.0 4.5 1700 15 ^(a)The values are thepercentage reduction in number of plaque per unit volume (well) whichare caused by viral lysis of the infected cells.

What is claimed is:
 1. A water-soluble zinc pyruvate of Formula I

where x is from 1.8 to 2.2 and n is from 0 to
 5. 2. The zinc pyruvateaccording to claim 1, wherein x is 2 and n is from 0 to
 3. 3. The zincpyruvate according to claim 1, wherein the pyruvate anion is2,2-dihydroxypropionate.
 4. The zinc pyruvate according to claim 1 insolid microcrystalline form.
 5. A process for preparing zinc pyruvateaccording to claim 1 comprising reacting a zinc salt of an organic acidor acidic organic keto compound or hydroxy compound with pyruvic acid ata temperature ranging from −20 to +90° C., optionally in the presence ofa solvent or diluent.
 6. The process according to claim 5, wherein thetemperature is from 10 to 50° C.
 7. The process according to claim 5,wherein the organic acid is selected from the group consisting of anamino-, keto- and hydroxy-carboxylic acids.
 8. The process according toclaim 5, wherein said zinc salt comprises an organic acid or acidicorganic keto compound or hydroxyl compound selected from the groupconsisting of formic acid, acetic acid, propionic acid, butyric acid,lactic acid, ascorbic acid, citric acid, tartaric acid, succinic acid,maleic acid, fulmaric acid, malic acid, aspartic acid, benzoic acid,glycolic acid, isovaleric acid, oleic acid, glycine and lysine.
 9. Theprocess according to claim 5, wherein said zinc salt of the organic acidor acidic organic keto compound or hydroxyl compound are anhydrous,hydrated or a moist product.
 10. The process according to claim 5,wherein the pyruvic acid is anhydrous, in an aqueous solution, ordissolved in a solvent or diluent.
 11. The process according to claim 5,wherein the solvent or diluent used is an organic solvent or water. 12.The process according to claim 11, wherein the organic solvent isselected from the group consisting of alcohols, ethers, ketones, esters,organic acids, aliphatic hydrocarbons and aromatic hydrocarbons.
 13. Theprocess of claim 5, wherein the pyruvic acid is formed as anintermediate by reacting alkali pyruvate with an inorganic acid attemperature of from −10 to +60° C.
 14. A pharmaceutical compositioncomprising the water-soluble zinc pyruvate of claim 1 and at least oneof a pharmaceutically acceptable carrier, diluent and adjuvant.
 15. Amethod of treating diabetes or treating colds inhibiting virus,enhancing cytroprotection, inducing micobicidal activity and absorbingradicals comprising administering an effective amount of the zincpyruvate of claim 1 to a subject in need thereof.
 16. A method for theprophylaxis or prevention of symptoms associated with zinc deficiencycomprising administering an effective amount of the zinc pyruvate ofclaim 1 to a subject in need thereof.
 17. A process for treating acondition selected from the group consisting of diabetes, colds, viralinfections, microbial infections and zinc deficiency comprisingadministering effective amount of a composition according to claim 14 toa subject in need thereof.
 18. A process for scavenging radicals compmging administering to a subject an effective amount of a compositionaccording to claim 14 to a subject.
 19. A process for inducingcytoprotection comprising administering to a subject an effective amountof a composition according to claim 14 to a subject.